The field of the invention is modulating angiogenesis by targeting a protein known as Kuz.
Genes of the ADAM family encode transmembrane proteins containing both metalloprotease and disintegrin domains (reviewed in Black and White, 1998 Curr.Opin.Cell Biol. 10, 654-659; Wolfsberg and White, 1996 Dev.Biol. 180, 389-401), and are involved in diverse biological processes in mammals such as fertilization (Cho et al., 1998 Science 281, 1857-1859), myoblast fusion (Yagami-Hiromasa et al., 1995 Nature 377, 652-656) and ectodomain shedding (Moss et al., 1997 Nature 385, 733-736; Black et al., 1997 Nature 385, 729-733; Peschon et al., 1998 Science 282, 1281-1284). The Drosophila kuzbanian (kuz) gene represents the first ADAM family member identified in invertebrates (Rooke et al., 1996 Science 273, 1227-1231). Previous genetic studies showed that kuz is required for lateral inhibition and axonal outgrowth during Drosophila neural development (Rooke et al., 1996; Fambrough et al., 1996 PNAS.USA 93, 13233-13238.; Pan and Rubin, 1997 Cell 90, 271-280; Sotillos et al., 1997 Development 124, 4769-4779). Specifically, during the lateral inhibition process, kuz acts upstream of Notch (Pan and Rubin, 1997; Sotillos et al., 1997), which encodes the transmembrane receptor for the lateral inhibition signal encoded by the Delta gene. More recently, a homolog of kuz was identified in C. elegans (SUP-17) that modulates the activity of a C. elegans homolog of Notch in a similar manner (Wen et al., 1997 Development 124, 4759-4767).
Vertebrate homologs of kuz have been isolated in Xenopus, bovine, mouse, rat and human. The bovine homolog of KUZ (also called MADM or ADAM 10) was initially isolated serendipitously based on its in vitro proteolytic activity on myelin basic protein, a cytolasmic protein that is unlikely the physiological substrate for the bovine KUZ protease (Howard et al., 1996 Biochem.J. 317, 45-50). In a recent study, we showed that expression of a dominant negative form of the murine kuz homolog (mkuz) in Xenopus leads to the generation of extra neurons, suggesting an evolutionarily conserved role for mkuz in regulating Notch signaling in vertebrate neurogenesis (Pan and Rubin, 1997). We have now generated mkuz-deficient mice using gene targeting in embryonic stem (ES) cells. We show that mkuz is essential for embryonic development. mkuz mutant mice die around embryonic day (E) 9.5, with severe defects in the nervous system, the paraxial mesoderm and the yolk sac vasculature. In the nervous system, mkuz mutant embryos show ectopic neuronal differentiation. In the paraxial mesoderm, mkuz mutant embryos show delayed and uncoordinated segmentation of the somites. These phenotypes are similar to those of mice lacking Notch-1 or components of the Notch pathway such as RBP-Jk (Conlon et al, 1995, Development 121, 1533-1545; Oka et al., 1995), indicating a conserved role for mkuz in modulating Notch signaling in mouse development. Furthermore, we detect no visible defect in Notch processing in our knockout animals. Besides the neurogenesis and somitogenesis defect, mkuz mutant mice also show severe defects in the yolk sac vasculature, with an enlarged and disordered capillary plexus and the absence of large vitelline vessels. Since such phenotype has not been observed in mice lacking Notch-l or RBP-Jk (Swiatek et al., 1994 Genes Dev 15, 707-719; Conlon et al, 1995; Oka et al., 1995 Development 121, 3291-3301), we determine that this phenotype reveals a novel function of mkuz that is distinct from its role in modulating Notch signaling. Taken together, our studies reveal the essential role for an ADAM family disintegrin metalloprotease in mammalian neurogenesis, somitogenesis and angiogenesis.
We disclosed that Kuz is involved in somitogenesis, neurogenesis and angiogenesis and provides a useful therapeutic target for intervention in associated pathologies. Accordingly, the invention provides methods and compositions relating to Kuz involvement in somitogenesis, neurogenesis, and particularly, angiogenesis. In one embodiment, the invention provides methods for modulating angiogenesis comprising the step of specifically modulating the activity of Kuz in a vertebrate animal predetermined to have a pathogenic angiogenesis. A wide variety of methods for specifically modulating Kuz activity are disclosed, including contacting the animal with an agent which specifically binds the Kuz or competes with the Kuz for substrate or a required cofactor.
In another embodiment, the invention provides methods for modulating angiogenesis comprising the steps of specifically modulating the activity of Kuz in a vertebrate animal not necessarily predetermined to have a pathogenic angiogenesis, but rather subsequently detecting a resultant angiogenic modulation in the animal.
The invention also provides methods for specifically detecting Kuz activity in a vertebrate animal predetermined to have a pathogenic angiogenesis; for example, using a KUZ specific protease assay or a KUZ specific immunobinding assay. The invention also provides methods for specifically detecting a pathogenic angiogenesis in a vertebrate animal having a predetermined Kuz activity; for example, by detecting a tumor associated with pathogenic angiogenesis.
The invention also provides methods for identifying a modulator of angiogenesis, comprising the steps of (a) contacting an angiogenic assay system comprising a predetermined amount of Kuz with a candidate agent, under conditions whereby but for the presence of the agent, the system provides a reference angiogenesis; and (b) detecting an agent-biased angiogenesis of the system; wherein a difference between the agent-biased angiogenesis and the reference angiogenesis indicates that the agent modulates angiogenesis in the system. Such methods may be embodied in an in vitro, cell based assay or an in vivo, animal-based assay.
The invention also provides kits and reagents adapted to the subject methods.
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d mean one or more, the term xe2x80x9corxe2x80x9d means and/or. Kuz refers to an art-recognized family of natural proteins which have been extensively described, encompassing natural orthologs and variants also well known in the art. For example, several forms of human KUZ have been described including WO98/37092 and WO97/31931; Mayer et al. (U.S. Pat. No. 5,922,546); and Rubin et al. (U.S. Pat. No. 5,935,792). Though often discussed and exemplified in terms of angiogenesis, the disclosed methods and reagents are to be understood to be generally applicable to pathogenic somitogenesis and neurogenesis as well.
Several disclosed applications involve specifically modulating the activity of Kuz in a vertebrate animal. A wide variety of methods for specifically modulating Kuz activity are disclosed, including contacting the animal with an agent which specifically binds the Kuz or competes with the Kuz for substrate or a required cofactor.
Agents which specifically bind kuz include metalloprotease inhibitors, such as hydroxamate metalloprotease inhibitors and TACE (TNF-alpha converting enzyme) inhibitors (for review, see Amour A, et al. Ann N Y Acad Sci 1999 Jun 30;878:728-31). Exemplary inhibitors include IC-3 (N-{D,L-[2-(hydroxyaminocaronyl)mehyl]-4-methyl-pentanoyl}-L-alanine, 2-aminoethyl amide, Black et al., Nature, 1997, Vol 385, 729-73; Galko and Tessier-Lavigne, Science, 2000, Vol 289, 1365-1367), GM6001 (NHOHCOCH2CH(I-Bu)CO-Trp-NHMe); GW9471 (see structure of GW9277, a biotinylated derivative of GW9471 used during the purification of TACE as shown in Moss et al, Nature, 1997, Vol 385, 733-736); and BB-94 (batimastat), a synthetic hydroxamate peptidomimetic matrix metalloproteinase inhibitor, see Hemandez-Pando R, et al. Int J Exp Pathol June;2000; 81(3):199-209. Useful natural MMP inhibitors include the tissue inhibitors of MMPs (TIMPs), such as TIMP-1 and TIMP-3 (see, e.g. Amour et al., FEBS Lett. May 2000 19;473(3):275-9).
Another class of inhibitors which specifically bind Kuz are polypeptides comprising immunoglobulin complementary determining regions (CDRs), particularly CDR3 regions which specifically bind Kuz. These encompass antibodies and antibody fragments such as F(ab) fragments. Methods for making and using therapeutic antibodies and antibody fragments are well known, e.g. U.S. Pat. No. 5,935,792.
Intracellular antibodies, or intrabodies, represent a class of neutralizing molecules with applications in gene therapy (vonMehren M,Weiner L M. (1996) Current Opinion in Oncology. 8: 493-498, Marasco Wash. (1997) Gene Therapy. 4: 11-15, Rondon I J, Marasco Wash. (1997) Annual Review of Microbiology. 51: 257-283). Anti-Kuz intrabodies are engineered single-chain antibodies in which the variable domain of the heavy chain is joined to the variable domain of the light chain through a peptide linker, preserving the affinity of the parent Kuz antibody (Rondon et al.). The anti-Kuz intrabodies are designed from either the polyclonal or monoclonal anti-Kuz antibody cDNA that encode antibodies that recognize the enzymatically active form of Kuz and which, upon binding, inhibit Kuz""s ability to transphosphorylate. Also, anti-Kuz intrabodies can be made from either polyclonal or monoclonal antibody cDNA that encodes an antibody that stimulates Kuz enzymatic activity. The anti-Kuz single chain intrabodies may be additionally modified with a C-terminal human C kappa domain to increase cytoplasmic stability and/or the C-terminal SV40 nuclear localization signal to direct the nascent intrabody to the nuclear compartment, respectively (Mhashilkar AM, et al. (1995) Embo Journal. 14: 1542-1551). In this regard, stably expressed single chain anti-Kuz intrabodies, and their modified forms, can be used to effectively target Kuz molecules either in the cytoplasm or nuclear compartments of eukaryotic cells.
The Kuz-specific intrabodies can be introduced into cultured cells by any one of several established methods that include the standard DNA transfection methods (Calcium phosphate, electrophoration, lipofectamine, etc.). The anti-Kuz intrabodies are first constructed into any one of a variety of inducible expression vectors tet repressible (Gossen M,Bujard H. (1992) Proc. Natl. Acad. Sci. USA. 89: 5547-5551) or IPTG inducible (Liu HS, et al. (1998) Biotechniques. 24: 624-632, Hannan G N, et al. (1993) Gene. 130: 233-239) or glucocorticoid inducible (using a GRE), constitutive expression vectors (such as CMV or RSV promoter driven vectors ) or tissue specific expression vectors using promoters of tissue specific expressed genes (such as the T cell receptor promoter). A key variation to express the anti-Kuz intrabodies tissues (as well as cell lines) is to construct appropriate viral expression vectors using standard protocols (Vile R G, et al.(1995) British Medical Bulletin. 51: 12-30, Shoji I, et al. (1997) J General Virology. 78: 2657-2664, Paulus W, et al (1996) J Virology. 70: 62-67). The anti-Kuz intrabody genes are substituted for the key viral genes and packaged into a viral particle by a host cell. The altered viral genome is integrated into the target tissue genome but is disrupted in a way that prevents the formation of new viral particles. Individual cells of the target tissues then produce the anti-Kuz intrabody transcripts and proteins.
A wide variety of agents may be used to specifically compete with Kuz for substrate or cofactors. Competitive inhibitors encompass numerous classes, including substituted hydroxamates, carboxylates, thiols, phosphonates, aminodiathiazols, and catechols which are know to inhibit Zn-metalloproteases through high-affinity zinc binding, and chelators of divalent cations, such as EDTA and 1,10-phenanthroline. Competitive inhibitors also include dominant negative Kuz mutants, wherein the protease domain is disrupted by deletion or point mutagenesis. Such Kuz mutants are known in the art and novel dominant negative mutants are readily made by targeted mutagenesis of residues within the protease domain followed by routine activity screening, see U.S. Pat. No.5,935,792. Exemplary dominant negative human kuz mutants are shown in Table 1.
xe2x80x9cNumberingxe2x80x9d refers to the amino acid residues as set forth in the human Kuz (SEQ ID NO:4) of U.S. Pat. No. 5,935,792. Corresponding mutations can be identified in other human Kuz proteins, such as disclosed in U.S. Pat. No. 5,922,546 and PCT publication WO 97/31931, by sequence alignment.
In a preferred embodiment, the dominant negative Kuz mutant is soluble, i.e. lacking the transmembrane domain but comprising one or more of the extracellular domains. Preferably, the soluble dominant negative mutant also lacks the signal peptide and prodomain, and comprises the cysteine-rich domain, the disintegrin domain and/or the metalloprotease domain. In another preferred embodiment, the soluble dominant negative mutant is fused to an unrelated polypeptide selected to facilitate purification, detection, or solubilization, or to provide some other function. Fusion proteins are generally produced by expressing a hybrid gene in which a nucleotide sequence encoding the soluble Kuz mutant joined in-frame to a nucleotide sequence encoding the selected unrelated polypeptide. A preferred unrelated protein is the constant (Fc) region of an immunoglobulin (e.g. a human IgG Fc region), which can render the resulting fusion protein more stable and with a longer half-life when used as a biotherapeutic.
Several disclosed applications involve a vertebrate animal, particularly a mouse, rat or human, which has been predetermined to have pathogenic somitogenesis, neurogenesis or particularly, angiogenesis. In other embodiments, the methods involve specifically detecting the pathogenic angiogenesis, somitogenesis or neurogenesis in the animal. Pathogenic angiogenesis for example, encompasses any condition presenting undesirably excessive or deficient angiogenesis, systemically or regionally; exemplary underlying conditions include cancer, diabetic retinopathy, rheumatoid arthritis, macular degeneration, psoriasis and other pathologies in which excessive, insufficient or misregulated angiogenesis plays a role. For example, our Kuz-deficient mice present upregulation of several neural specific genes, including Mash-1 and neurogenin, indicating an excess of neural precursors. These mice also present defective somitogenesis as revealed by loss of D111 expression in somites and severe phenotypic disruption of the somites. In addition, the mice present pathogenic angiogenesis, wherein vitelline vessels in the embryonic yolk sack fail to develop. The pathogenic somitogenesis, neurogenesis or angiogenesis are readily detected by routine methods, such as histological exam, expression of correlating marker genes, etc. In addition, numerous in vitro model systems are known, such as endothelial cell based angiogenesis assays, as exemplified below. In many cases, detection is effected inferentially by detecting a condition, such as a tumor, which is associated with a pathogenic angiogenesis. Angiogenesis in particular is detected by any convenient means, including in vitro, cell-based assays such as huvec assays and in vivo measures such as blood flow paramenters, microvessel density, vascular endothelial growth factor levels (see, e.g. Lee et al. Obstet Gyneco October;2000 96(4):615-21), growth factor receptors (e.g. Shin et al. 2000 J Cancer Rec Clin Oncol 126, 519-28. etc., These assays may be practiced in model systems, such as heterologous transplant systems, e.g. Rofstad et al. 2000 Cancer Res 60, 4932-8.
The present disclosure that Kuz provides a useful therapeutic target for conditions associated with pathogenic somitogenesis, neurogenesis or angiogenesis provides numerous applications that will be apparent to those skilled in the artxe2x80x94any application premised on the used of Kuz as a therapeutic target for conditions associated with pathogenic somitogenesis, neurogenesis or angiogenesis. For example, in one embodiment, the invention provides methods for modulating angiogenesis comprising the steps of specifically modulating the activity of Kuz in a vertebrate animal not necessarily predetermined to have a pathogenic angiogenesis, but rather subsequently detecting a resultant angiogenic modulation in the animal. In another embodiment, the invention also provides methods for specifically detecting Kuz activity in a vertebrate animal predetermined to have a pathogenic angiogenesis; for example, using a KUZ specific protease assay or a KUZ specific immunobinding assay. In another embodiment, the invention provides methods for specifically detecting a pathogenic angiogenesis in a vertebrate animal having a predetermined Kuz activity; for example, by detecting a tumor associated with pathogenic angiogenesis.
The invention also provides methods for identifying a modulator of angiogenesis which is a priori known to be associated with Kuz activity. An exemplary such method comprises the steps of (a) contacting an angiogenic assay system comprising a predetermined amount of Kuz with a candidate agent, under conditions whereby but for the presence of the agent, the system provides a reference angiogenesis; and (b) detecting an agent-biased angiogenesis of the system; wherein a difference between the agent-biased angiogenesis and the reference angiogenesis indicates that the agent modulates angiogenesis in the system. Such screening methods may be embodied in an in vitro, cell based assay or an in vivo, animal-based assays, such as described below.
Without further description, one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.